The Respiratory Chain & Oxidative Phosphorylation

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The Respiratory Chain & Oxidative Phosphorylation 12

Peter A. Mayes, PhD, DSc, & Kathleen M. Botham, PhD, DSc

BIOMEDICAL IMPORTANCE trapping the liberated free energy as high-energy phosphate, and the enzymes of β-oxidation and of the citric Aerobic organisms are able to capture a far greater pro- acid cycle (Chapters 22 and 16) that produce most of portion of the available free energy of respiratory sub- the reducing equivalents. strates than anaerobic organisms. Most of this takes place inside mitochondria, which have been termed the “powerhouses” of the cell. Respiration is coupled to the Components of the Respiratory Chain generation of the high-energy intermediate, ATP, by Are Arranged in Order of Increasing oxidative phosphorylation, and the chemiosmotic Redox Potential theory offers insight into how this is accomplished. A number of drugs (eg, amobarbital) and poisons (eg, Hydrogen and electrons flow through the respiratory chain (Figure 12–3) through a redox span of 1.1 V cyanide, carbon monoxide) inhibit oxidative phos- + phorylation, usually with fatal consequences. Several in- from NAD /NADH to O2/2H2O (Table 11–1). The herited defects of mitochondria involving components respiratory chain consists of a number of redox carriers of the respiratory chain and oxidative phosphorylation that proceed from the NAD-linked dehydrogenase sys- have been reported. Patients present with myopathy tems, through flavoproteins and cytochromes, to mole- and encephalopathy and often have lactic acidosis. cular oxygen. Not all substrates are linked to the respiratory chain through NAD-specific dehydrogenases; some, because their redox potentials are more positive SPECIFIC ENZYMES ACT AS MARKERS (eg, fumarate/succinate; Table 11–1), are linked directly to flavoprotein dehydrogenases, which in turn are OF COMPARTMENTS SEPARATED BY linked to the cytochromes of the respiratory chain (Fig- THE MITOCHONDRIAL MEMBRANES ure 12–4). Mitochondria have an outer membrane that is perme- Ubiquinone or Q (coenzyme Q) (Figure 12–5) able to most metabolites, an inner membrane that is links the flavoproteins to cytochrome b, the member of selectively permeable, and a matrix within (Figure the cytochrome chain of lowest redox potential. Q ex- 12–1). The outer membrane is characterized by the ists in the oxidized quinone or reduced quinol form presence of various enzymes, including acyl-CoA syn- under aerobic or anaerobic conditions, respectively. thetase and glycerolphosphate acyltransferase. Adenylyl The structure of Q is very similar to that of vitamin K kinase and creatine kinase are found in the intermem- and vitamin E (Chapter 45) and of plastoquinone, brane space. The phospholipid cardiolipin is concen- found in chloroplasts. Q acts as a mobile component of trated in the inner membrane together with the en- the respiratory chain that collects reducing equivalents zymes of the respiratory chain. from the more fixed flavoprotein complexes and passes them on to the cytochromes. An additional component is the iron-sulfur protein THE RESPIRATORY CHAIN COLLECTS (FeS; nonheme iron) (Figure 12–6). It is associated & OXIDIZES REDUCING EQUIVALENTS with the flavoproteins (metalloflavoproteins) and with cytochrome b. The sulfur and iron are thought to take Most of the energy liberated during the oxidation of part in the oxidoreduction mechanism between flavin carbohydrate, fatty acids, and amino acids is made and Q, which involves only a single e− change, the iron available within mitochondria as reducing equivalents atom undergoing oxidoreduction between Fe2+ and (H or electrons) (Figure 12–2). Mitochondria con- Fe3+. tain the respiratory chain, which collects and trans- Pyruvate and α-ketoglutarate dehydrogenase have ports reducing equivalents directing them to their final complex systems involving lipoate and FAD prior to reaction with oxygen to form water, the machinery for the passage of electrons to NAD, while electron trans- 92 ch12.qxd 2/13/2003 2:46 PM Page 93

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Electrons flow from Q through the series of cytochromes in order of increasing redox potential to molecular oxygen (Figure 12–4). The terminal cytochrome aa3 (cytochrome oxidase), responsible for the final com- Phosphorylating bination of reducing equivalents with molecular oxy- complexes gen, has a very high affinity for oxygen, allowing the respiratory chain to function at maximum rate until the tissue has become depleted of O2. Since this is an irre- MATRIX versible reaction (the only one in the chain), it gives direction to the movement of reducing equivalents and to the production of ATP, to which it is coupled. Functionally and structurally, the components of Cristae the respiratory chain are present in the inner mitochondrial membrane as four protein-lipid respiratory chain complexes that span the membrane. Cytochrome c is the only soluble cytochrome and, together with Q, INNER seems to be a more mobile component of the respira- MEMBRANE tory chain connecting the fixed complexes (Figures 12–7 and 12–8). OUTER MEMBRANE THE RESPIRATORY CHAIN PROVIDES MOST OF THE ENERGY CAPTURED DURING CATABOLISM ADP captures, in the form of high-energy phosphate, a Figure 12–1. Structure of the mitochondrial mem- significant proportion of the free energy released by branes. Note that the inner membrane contains many catabolic processes. The resulting ATP has been called folds, or cristae. the energy “currency” of the cell because it passes on this free energy to drive those processes requiring energy (Figure 10–6). fers from other dehydrogenases, eg, L(+)-3-hydroxyacyl- There is a net direct capture of two high-energy CoA dehydrogenase, couple directly with NAD. phosphate groups in the glycolytic reactions (Table The reduced NADH of the respiratory chain is in 17–1), equivalent to approximately 103.2 kJ/mol of turn oxidized by a metalloflavoprotein enzyme—NADH glucose. (In vivo, ∆G for the synthesis of ATP from dehydrogenase. This enzyme contains FeS and FMN, ADP has been calculated as approximately 51.6 kJ/mol. is tightly bound to the respiratory chain, and passes re- (It is greater than ∆G0′ for the hydrolysis of ATP as ducing equivalents on to Q. given in Table 10–1, which is obtained under standard

FOOD ATP Fat Fatty acids + β-Oxidation Glycerol O2 Citric Carbohydrate Glucose, etc acid Acetyl – CoA 2H H2O cycle

Respiratory chain Protein Amino acids Digestion and absorption MITOCHONDRION ADP Extramitochondrial sources of reducing equivalents

Figure 12–2. Role of the respiratory chain of mitochondria in the conversion of food energy to ATP. Oxidation of the major foodstuffs leads to the generation of reducing equivalents (2H) that are collected by the respiratory chain for oxidation and coupled generation of ATP. ch12.qxd 2/13/2003 2:46 PM Page 94

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+ 3+ AH2 NAD FpH2 2Fe H2O

Substrate Flavoprotein Cytochromes

2+ 1 Figure 12–3. Transport of reducing A NADH Fp 2Fe /2O 2 equivalents through the respiratory H+ H+ 2H+ 2H+ chain.

concentrations of 1.0 mol/L.) Since 1 mol of glucose dent that the respiratory chain is responsible for a large yields approximately 2870 kJ on complete combustion, proportion of total ATP formation. the energy captured by phosphorylation in glycolysis is small. Two more high-energy phosphates per mole of Respiratory Control Ensures glucose are captured in the citric acid cycle during the a Constant Supply of ATP conversion of succinyl CoA to succinate. All of these phosphorylations occur at the substrate level. When The rate of respiration of mitochondria can be con- substrates are oxidized via an NAD-linked dehydrogen- trolled by the availability of ADP. This is because oxi- ase and the respiratory chain, approximately 3 mol of dation and phosphorylation are tightly coupled; ie, oxi- inorganic phosphate are incorporated into 3 mol of dation cannot proceed via the respiratory chain without ADP to form 3 mol of ATP per half mol of O2 con- concomitant phosphorylation of ADP. Table 12–1 sumed; ie, the P:O ratio = 3 (Figure 12–7). On the shows the five conditions controlling the rate of respira- other hand, when a substrate is oxidized via a flavopro- tion in mitochondria. Most cells in the resting state are tein-linked dehydrogenase, only 2 mol of ATP are in state 4, and respiration is controlled by the availabil- formed; ie, P:O = 2. These reactions are known as ox- ity of ADP. When work is performed, ATP is con- idative phosphorylation at the respiratory chain verted to ADP, allowing more respiration to occur, level. Such dehydrogenations plus phosphorylations at which in turn replenishes the store of ATP. Under cer- the substrate level can now account for 68% of the free tain conditions, the concentration of inorganic phos- energy resulting from the combustion of glucose, cap- phate can also affect the rate of functioning of the respi- tured in the form of high-energy phosphate. It is evi- ratory chain. As respiration increases (as in exercise),

Succinate Choline Proline 3-Hydroxyacyl-CoA 3-Hydroxybutyrate Glutamate Fp Malate (FAD) Pyruvate Isocitrate FeS

Fp Lipoate Fp NAD (FMN) Q Cyt b Cyt c1 Cyt c Cyt aa3 O2 (FAD) FeS FeS Cu

α -Ketoglutarate Fp FeS (FAD) ETF FeS (FAD)

Fp FeS: Iron-sulfur protein (FAD) ETF: Electron-transferring flavoprotein Fp: Flavoprotein Q: Ubiquinone Glycerol 3-phosphate Acyl-CoA Cyt: Cytochrome Sarcosine Dimethylglycine

Figure 12–4. Components of the respiratory chain in mitochondria, showing the collecting points for reduc-

ing equivalents from important substrates. FeS occurs in the sequences on the O2 side of Fp or Cyt b. ch12.qxd 2/13/2003 2:46 PM Page 95

THE RESPIRATORY CHAIN & OXIDATIVE PHOSPHORYLATION /95

O H OH H OH (H+ + e–) (H+ + e–)

CH3O CH3 CH3

CH3O [CH2CH CCH2]nH

O •O OH Fully oxidized or Semiquinone form Reduced or quinol form quinone form (free radical) (hydroquinone)

Figure 12–5. Structure of ubiquinone (Q). n = Number of isoprenoid units, which is

10 in higher animals, ie, Q10.

the cell approaches state 3 or state 5 when either the ca- MANY POISONS INHIBIT THE pacity of the respiratory chain becomes saturated or the RESPIRATORY CHAIN PO2 decreases below the Km for cytochrome a3. There is also the possibility that the ADP/ATP transporter (Fig- Much information about the respiratory chain has been ure 12–9), which facilitates entry of cytosolic ADP into obtained by the use of inhibitors, and, conversely, this and ATP out of the mitochondrion, becomes rate- has provided knowledge about the mechanism of action limiting. of several poisons (Figure 12–7). They may be classified Thus, the manner in which biologic oxidative as inhibitors of the respiratory chain, inhibitors of ox- processes allow the free energy resulting from the oxida- idative phosphorylation, and uncouplers of oxidative tion of foodstuffs to become available and to be cap- phosphorylation. tured is stepwise, efficient (approximately 68%), and Barbiturates such as amobarbital inhibit NAD- controlled—rather than explosive, inefficient, and un- linked dehydrogenases by blocking the transfer from controlled, as in many nonbiologic processes. The re- FeS to Q. At sufficient dosage, they are fatal in vivo. maining free energy that is not captured as high-energy Antimycin A and dimercaprol inhibit the respiratory phosphate is liberated as heat. This need not be consid- chain between cytochrome b and cytochrome c. The ered “wasted,” since it ensures that the respiratory sys- classic poisons H2S, carbon monoxide, and cyanide tem as a whole is sufficiently exergonic to be removed inhibit cytochrome oxidase and can therefore totally ar- from equilibrium, allowing continuous unidirectional rest respiration. Malonate is a competitive inhibitor of flow and constant provision of ATP. It also contributes succinate dehydrogenase. to maintenance of body temperature. Atractyloside inhibits oxidative phosphorylation by inhibiting the transporter of ADP into and ATP out of the mitochondrion (Figure 12–10). Pr The action of uncouplers is to dissociate oxidation Cys in the respiratory chain from phosphorylation. These compounds are toxic in vivo, causing respiration to be- S come uncontrolled, since the rate is no longer limited S Fe by the concentration of ADP or Pi. The uncoupler that has been used most frequently is 2,4-dinitrophenol, Pr Cys S Fe S but other compounds act in a similar manner. The an- tibiotic oligomycin completely blocks oxidation and phosphorylation by acting on a step in phosphorylation Fe S (Figures 12–7 and 12–8). S Cys S Fe THE CHEMIOSMOTIC THEORY EXPLAINS Pr S THE MECHANISM OF OXIDATIVE Cys PHOSPHORYLATION Pr Mitchell’s chemiosmotic theory postulates that the energy from oxidation of components in the respiratory S Figure 12–6. Iron-sulfur-protein complex (Fe4S4). , chain is coupled to the translocation of hydrogen ions acid-labile sulfur; Pr, apoprotein; Cys, cysteine residue. (protons, H+) from the inside to the outside of the Some iron-sulfur proteins contain two iron atoms and inner mitochondrial membrane. The electrochemical two sulfur atoms (Fe2S2). potential difference resulting from the asymmetric dis- ch12.qxd 2/13/2003 2:46 PM Page 96

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Malonate Complex II

FAD Succinate FeS – Carboxin TTFA H2S CO BAL – – Antimycin A CN – Complex IV Complex I Complex III Cyt a Cyt a3 NADH FMN, FeS Q Cyt b, FeS, Cyt c1 Cyt c O2 Cu Cu – – Piericidin A Uncouplers – Amobarbital – Uncouplers – Rotenone

Oligomycin – – Oligomycin –

ADP + Pi ATP ADP + Pi ATP ADP + Pi ATP

Figure 12–7. Proposed sites of inhibition (− ) of the respiratory chain by specific drugs, chemicals, and antibi- otics. The sites that appear to support phosphorylation are indicated. BAL, dimercaprol. TTFA, an Fe-chelating agent. Complex I, NADH:ubiquinone oxidoreductase; complex II, succinate:ubiquinone oxidoreductase; complex III, ubiquinol:ferricytochrome c oxidoreductase; complex IV, ferrocytochrome c:oxygen oxidoreductase. Other ab- breviations as in Figure 12–4.

tribution of the hydrogen ions is used to drive the units are attached to a membrane protein complex mechanism responsible for the formation of ATP (Fig- known as F0, which also consists of several protein sub- ure 12–8). units. F0 spans the membrane and forms the proton channel. The flow of protons through F0 causes it to rotate, driving the production of ATP in the F1 complex The Respiratory Chain Is a Proton Pump (Figure 12–9). Estimates suggest that for each NADH Each of the respiratory chain complexes I, III, and IV oxidized, complex I translocates four protons and com- (Figures 12–7 and 12–8) acts as a proton pump. The plexes III and IV translocate 6 between them. As four inner membrane is impermeable to ions in general but protons are taken into the mitochondrion for each ATP particularly to protons, which accumulate outside the exported, the P:O ratio would not necessarily be a com- membrane, creating an electrochemical potential dif- plete integer, ie, 3, but possibly 2.5. However, for sim- ∆µ + plicity, a value of 3 for the oxidation of NADH + H+ ference across the membrane ( H ).This consists of a chemical potential (difference in pH) and an electrical and 2 for the oxidation of FADH2 will continue to be potential. used throughout this text. Experimental Findings Support A Membrane-Located ATP Synthase the Chemiosmotic Theory Functions as a Rotary Motor to Form ATP (1) Addition of protons (acid) to the external The electrochemical potential difference is used to drive medium of intact mitochondria leads to the generation a membrane-located ATP synthase which in the pres- of ATP. ence of Pi + ADP forms ATP (Figure 12–8). Scattered (2) Oxidative phosphorylation does not occur in solu- over the surface of the inner membrane are the phos- ble systems where there is no possibility of a vectorial phorylating complexes, ATP synthase, responsible for ATP synthase. A closed membrane must be present in the production of ATP (Figure 12–1). These consist of order to achieve oxidative phosphorylation (Figure 12–8). several protein subunits, collectively known as F1, (3) The respiratory chain contains components or- which project into the matrix and which contain the ganized in a sided manner (transverse asymmetry) as re- phosphorylation mechanism (Figure 12–8). These sub- quired by the chemiosmotic theory. ch12.qxd 2/13/2003 2:46 PM Page 97

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H+ Pro ton cir cu Oligomycin it – F Phospholipid 0 bilayer

F1 H+ I ATP SYNTHASE NADH + H+

ADP + Pi ATP + H2O + Q t NAD ra Mitochondrial n P Respiratory s r III o inner (coupling) l + o

t + (electron o

H c H a

membrane n transport)

o n chain 1 2 + / O2 H C

H O IV Uncoupling agents 2 + H INSIDE H+ pH gradient (∆pH) – Electrical potential

OUTSIDE + (∆Ψ) Figure 12–8. Principles of the chemiosmotic theory of oxidative phosphorylation. The main proton circuit is created by the coupling of oxidation in the respiratory chain to proton translocation from the inside to the outside of the membrane, driven by the respiratory chain complexes I, III, and IV, each of which acts as a pro-

ton pump. Q, ubiquinone; C, cytochrome c; F1, F0, protein subunits which utilize energy from the proton gradient to promote phosphorylation. Uncoupling agents such as dinitrophenol allow leakage of H+ across the membrane, thus collapsing the electrochemical proton gradient. Oligomycin specifically blocks conduction + of H through F0.

The Chemiosmotic Theory Can Account for Respiratory Control and the Action of Uncouplers The electrochemical potential difference across the membrane, once established as a result of proton transloca- Table 12–1. States of respiratory control. tion, inhibits further transport of reducing equivalents through the respiratory chain unless discharged by back- Conditions Limiting the Rate of Respiration translocation of protons across the membrane through the vectorial ATP synthase. This in turn depends on State 1 Availability of ADP and substrate availability of ADP and Pi. State 2 Availability of substrate only Uncouplers (eg, dinitrophenol) are amphipathic State 3 The capacity of the respiratory chain itself, when (Chapter 14) and increase the permeability of the lipoid all substrates and components are present in inner mitochondrial membrane to protons (Figure saturating amounts 12–8), thus reducing the electrochemical potential and State 4 Availability of ADP only short-circuiting the ATP synthase. In this way, oxida- State 5 Availability of oxygen only tion can proceed without phosphorylation. 5475ch12.qxd_ccI 2/26/03 8:01 AM Page 98

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β ing electrical and osmotic equilibrium. The inner ATP bilipoid mitochondrial membrane is freely permeable α α to uncharged small molecules, such as oxygen, water, γ ADP ββ CO2, and NH3, and to monocarboxylic acids, such as + α ATP 3-hydroxybutyric, acetoacetic, and acetic. Long-chain Pi fatty acids are transported into mitochondria via the carnitine system (Figure 22–1), and there is also a special carrier for pyruvate involving a symport that utilizes the H+ gradient from outside to inside the mitochondrion (Figure 12–10). However, dicarboxylate and tri-

γ H+

Inside Inner OUTSIDE mitochondrial INSIDE membrane Mitochondrial inner N-Ethylmaleimide membrane OH– C C 1 Outside C – C H2PO4 CC N-Ethylmaleimide – Hydroxycinnamate Pyruvate– 2 + + H H – 2– Figure 12–9. Mechanism of ATP production by ATP HPO4 synthase. The enzyme complex consists of an F0 sub- 3 complex which is a disk of “C” protein subunits. At- Malate2– tached is a γ-subunit in the form of a “bent axle.” Pro- tons passing through the disk of “C” units cause it and Malate2– γ γ 4 the attached -subunit to rotate. The -subunit fits in- 3– α β Citrate side the F1 subcomplex of three - and three -sub- + H+ units, which are fixed to the membrane and do not ro- Malate2– tate. ADP and Pi are taken up sequentially by the β-subunits to form ATP, which is expelled as the rotat- 5 2– γ β α-Ketoglutarate ing -subunit squeezes each -subunit in turn. Thus, – three ATP molecules are generated per revolution. For 3– clarity, not all the subunits that have been identified are ADP shown—eg, the “axle” also contains an ε-subunit. 6 ATP4– Atractyloside

Figure 12–10. Transporter systems in the inner mi- THE RELATIVE IMPERMEABILITY tochondrial membrane. 1 , phosphate transporter; OF THE INNER MITOCHONDRIAL 2 , pyruvate symport; 3 , dicarboxylate transporter; 4 5 α MEMBRANE NECESSITATES , tricarboxylate transporter; , -ketoglutarate transporter; 6 , adenine nucleotide transporter. N-Ethyl- EXCHANGE TRANSPORTERS maleimide, hydroxycinnamate, and atractyloside inhibit Exchange diffusion systems are present in the mem- (− ) the indicated systems. Also present (but not brane for exchange of anions against OH− ions and shown) are transporter systems for glutamate/aspar- cations against H+ ions. Such systems are necessary for tate (Figure 12–13), glutamine, ornithine, neutral amino uptake and output of ionized metabolites while preserv- acids, and carnitine (Figure 22–1). ch12.qxd 2/13/2003 2:46 PM Page 99

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carboxylate anions and amino acids require specific Ionophores Permit Specific Cations transporter or carrier systems to facilitate their passage to Penetrate Membranes across the membrane. Monocarboxylic acids penetrate more readily in their undissociated and more lipid-solu- Ionophores are lipophilic molecules that complex specific cations and facilitate their transport through bio- ble form. + The transport of di- and tricarboxylate anions is logic membranes, eg, valinomycin (K ). The classic closely linked to that of inorganic phosphate, which uncouplers such as dinitrophenol are, in fact, proton − ionophores. penetrates readily as the H2PO4 ion in exchange for OH−. The net uptake of malate by the dicarboxylate transporter requires inorganic phosphate for exchange A Proton-Translocating Transhydrogenase in the opposite direction. The net uptake of citrate, Is a Source of Intramitochondrial NADPH isocitrate, or cis-aconitate by the tricarboxylate trans- Energy-linked transhydrogenase, a protein in the inner porter requires malate in exchange. α-Ketoglutarate mitochondrial membrane, couples the passage of pro- transport also requires an exchange with malate. The tons down the electrochemical gradient from outside to adenine nucleotide transporter allows the exchange of inside the mitochondrion with the transfer of H from ATP and ADP but not AMP. It is vital in allowing intramitochondrial NADH to NADPH for intramito- ATP exit from mitochondria to the sites of extramitochondrial enzymes such as glutamate dehydrogenase chondrial utilization and in allowing the return of ADP and hydroxylases involved in steroid synthesis. for ATP production within the mitochondrion (Figure 12–11). Na+ can be exchanged for H+, driven by the proton gradient. It is believed that active uptake of Ca2+ Oxidation of Extramitochondrial NADH by mitochondria occurs with a net charge transfer of 1 Is Mediated by Substrate Shuttles + 2+ + (Ca uniport), possibly through a Ca /H antiport. NADH cannot penetrate the mitochondrial mem- Calcium release from mitochondria is facilitated by ex- brane, but it is produced continuously in the cytosol by + change with Na . 3-phosphoglyceraldehyde dehydrogenase, an enzyme in the glycolysis sequence (Figure 17–2). However, under aerobic conditions, extramitochondrial NADH does not accumulate and is presumed to be oxidized by the respiratory chain in mitochondria. The transfer of reducing Inner equivalents through the mitochondrial membrane re- OUTSIDEmitochondrial INSIDE quires substrate pairs, linked by suitable dehydrogen- membrane ases on each side of the mitochondrial membrane. The

F1 mechanism of transfer using the glycerophosphate ATP SYNTHASE shuttle is shown in Figure 12–12). Since the mitochon-

+ drial enzyme is linked to the respiratory chain via a 3H flavoprotein rather than NAD, only 2 mol rather than 3 mol of ATP are formed per atom of oxygen con- sumed. Although this shuttle is present in some tissues ATP 4– (eg, brain, white muscle), in others (eg, heart muscle) it is deficient. It is therefore believed that the malate shuttle system (Figure 12–13) is of more universal 2 ADP3– utility. The complexity of this system is due to the impermeability of the mitochondrial membrane to oxalo- – Pi acetate, which must react with glutamate and transami- 1 nate to aspartate and α-ketoglutarate before transport H+ through the mitochondrial membrane and reconstitu- Figure 12–11. Combination of phosphate trans- tion to oxaloacetate in the cytosol. porter (1 ) with the adenine nucleotide transporter (2 ) + Ion Transport in Mitochondria in ATP synthesis. The H /Pi symport shown is equiva- − Is Energy-Linked lent to the Pi/OH antiport shown in Figure 12–10. Four protons are taken into the mitochondrion for each ATP Mitochondria maintain or accumulate cations such as + + 2+ 2+ exported. However, one less proton would be taken in K , Na , Ca , and Mg , and Pi. It is assumed that a when ATP is used inside the mitochondrion. primary proton pump drives cation exchange. ch12.qxd 2/13/2003 2:46 PM Page 100

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OUTER INNER MEMBRANE MEMBRANE

CYTOSOL MITOCHONDRION

NAD+ Glycerol 3-phosphate Glycerol 3-phosphate FAD

GLYCEROL-3-PHOSPHATE GLYCEROL-3-PHOSPHATE DEHYDROGENASE DEHYDROGENASE (CYTOSOLIC) (MITOCHONDRIAL) + NADH + H Dihydroxyacetone Dihydroxyacetone FADH2 phosphate phosphate

Respiratory chain

Figure 12–12. Glycerophosphate shuttle for transfer of reducing equivalents from the cytosol into the mitochondrion.

The Creatine Phosphate Shuttle ported into the cytosol via protein pores in the outer Facilitates Transport of High-Energy mitochondrial membrane, becoming available for gen- Phosphate From Mitochondria eration of extramitochondrial ATP. This shuttle (Figure 12–14) augments the functions of CLINICAL ASPECTS creatine phosphate as an energy buffer by acting as a dynamic system for transfer of high-energy phosphate The condition known as fatal infantile mitochondrial from mitochondria in active tissues such as heart and myopathy and renal dysfunction involves severe dim- skeletal muscle. An isoenzyme of creatine kinase (CKm) inution or absence of most oxidoreductases of the respi- is found in the mitochondrial intermembrane space, ratory chain. MELAS (mitochondrial encephalopathy, catalyzing the transfer of high-energy phosphate to cre- lactic acidosis, and stroke) is an inherited condition due atine from ATP emerging from the adenine nucleotide to NADH:ubiquinone oxidoreductase (complex I) or transporter. In turn, the creatine phosphate is trans- cytochrome oxidase deficiency. It is caused by a muta-

INNER CYTOSOL MEMBRANE MITOCHONDRION NAD+ Malate Malate NAD+ 1 MALATE DEHYDROGENASE MALATE DEHYDROGENASE

NADH Oxaloacetate α-KG α-KG Oxaloacetate NADH + H+ + H+

TRANSAMINASE TRANSAMINASE

GlutamateAsp Asp Glutamate

H+ H+

Figure 12–13. Malate shuttle for transfer of reducing equivalents from the cytosol into the mitochondrion. 1 Ketoglutarate transporter; 2 , glutamate/aspartate transporter (note the proton symport with glutamate). 5475ch12.qxd_ccI 2/26/03 8:01 AM Page 101

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Energy-requiring processes (eg, muscle contraction) ATP ADP

CKa

ATP ADP

Creatine Creatine-P

CKc

CKg ATP ADP

Glycolysis Cytosol Outer mitochondrial b mem rane P Figure 12–14. The creatine phosphate shuttle of P heart and skeletal muscle. The shuttle allows rapid CKm transport of high-energy phosphate from the mito-

chondrial matrix into the cytosol. CKa, creatine kinase Inter-membrane concerned with large requirements for ATP, eg, mus- ATP ADP space

cular contraction; CKc, creatine kinase for maintaining Adenine equilibrium between creatine and creatine phosphate nucleotide and ATP/ADP; CK , creatine kinase coupling glycolysis transporter m g i In to n m ch e to creatine phosphate synthesis; CKm, mitochondrial e o r m n b d creatine kinase mediating creatine phosphate produc- Oxidative ra r n ia tion from ATP formed in oxidative phosphorylation; P, phosphorylation e l pore protein in outer mitochondrial membrane. Matrix

tion in mitochondrial DNA and may be involved in • Because the inner mitochondrial membrane is imper- Alzheimer’s disease and diabetes mellitus. A number of meable to protons and other ions, special exchange drugs and poisons act by inhibition of oxidative phos- transporters span the membrane to allow passage of – − 4− 3− phorylation (see above). ions such as OH , Pi , ATP , ADP , and metabolites, without discharging the electrochemical gradient across the membrane. SUMMARY • Many well-known poisons such as cyanide arrest respiration by inhibition of the respiratory chain. • Virtually all energy released from the oxidation of carbohydrate, fat, and protein is made available in − REFERENCES mitochondria as reducing equivalents (H or e ). These are funneled into the respiratory chain, where Balaban RS: Regulation of oxidative phosphorylation in the mam- they are passed down a redox gradient of carriers to malian cell. Am J Physiol 1990;258:C377. their final reaction with oxygen to form water. Hinkle PC et al: Mechanistic stoichiometry of mitochondrial ox- • The redox carriers are grouped into respiratory chain idative phosphorylation. Biochemistry 1991;30:3576. complexes in the inner mitochondrial membrane. Mitchell P: Keilin’s respiratory chain concept and its chemiosmotic These use the energy released in the redox gradient to consequences. Science 1979;206:1148. pump protons to the outside of the membrane, creat- Smeitink J et al: The genetics and pathology of oxidative phosphor- ing an electrochemical potential across the membrane. ylation. Nat Rev Genet 2001;2:342. Tyler DD: The Mitochondrion in Health and Disease. VCH Pub- • Spanning the membrane are ATP synthase com- lishers, 1992. plexes that use the potential energy of the proton gra- Wallace DC: Mitochondrial DNA in aging and disease. Sci Am dient to synthesize ATP from ADP and Pi. In this 1997;277(2):22. way, oxidation is closely coupled to phosphorylation Yoshida M et al: ATP synthase—a marvellous rotary engine of the to meet the energy needs of the cell. cell. Nat Rev Mol Cell Biol 2001;2:669.